Network pharmacology combined with experimental verification to explore the potential mechanism of naringenin in the treatment of cervical cancer

Cervical cancer is the second leading cause of morbidity and mortality in women worldwide. Traditional treatment methods have become limited. Naringenin, a flavonoid abundant in various fruits and herbal medicines, has demonstrated anti-tumor properties among other effects. This research undertook to elucidate the mechanism of naringenin in the context of cervical cancer treatment by leveraging network pharmacology and performing experimental validation. Initial steps involved predicting potential naringenin targets and subsequently screening for overlaps between these targets and those related to cervical cancer, followed by analysis of their interrelationships. Molecular docking was subsequently utilized to verify the binding effect of the central target. Within the framework of network pharmacology, it was discovered that naringenin might possess anti-cancer properties specific to cervical cancer. Following this, the anti-tumor effects of naringenin on Hela cell viability, migration, and invasion were assessed employing CCK-8, transwell, wound healing assays, and western blotting. Experimental data indicated that naringenin attenuates the migration and invasion of Hela cells via downregulation EGFR/PI3K/AKT signaling pathway. Thus, our findings suggest that naringenin has therapeutic impacts on cervical cancer via multiple mechanisms, primarily by inhibiting the migration and invasion through the EGFR/PI3K/AKT/mTOR pathway. This study offers fresh insights for future clinical studies.


Visual function enrichment analysis
The naringenin and cervical cancer targets were imported into Metascape for GO (Cellular Component, Molecular Function, Biological Process) and KEGG pathway enrichment analysis.The top 20 biological functions and pathways were selected according to gene enrichment and depicted in a histogram or bubble chart.

Molecule docking identification
Molecular docking technology was harnessed to examine the binding effects of hub targets within the compound.The experimental procedure has previously been described 22 .After the 3D structure of naringenin was optimized, the corresponding 3D structure of proteins corresponding to the hub targets was procured from the PDB database.AutoDock Tools were used for hydrogenation, active pocket coordinates determination, and parameter setting.AutoDock Vina subsequently conducted molecular docking and recorded the binding energy.

Cell culture and treatment
The HeLa cell line was obtained from the National Cell Bank of China (NCBC, Shanghai).The cells were cultivated in DMEM (Gibco, USA), supplemented with 10% FBS (Gibco, USA), 100 units/ml penicillin, and 100 mg/ ml streptomycin (Beyotime, China).Culturing was conducted under standard conditions (5% CO 2 , 37 °C, 95% humidity), with cells consistently maintained in the growth phase.Naringenin (Macklin, China) was dissolved in DMSO (Gibco, USA) to create a 50 mM stock solution, which was then stored at − 20 °C.Treatments were administered to cells with and without naringenin at concentrations of 0 µM, 100 µM, and 200 µM.

Cell viability assay
HeLa cells, representative of cervical cancer, were seeded into 96-well plates and exposed to varying concentrations of naringenin (0 μM, 100 μM, 200 μM) for 24 and 48 h at 37 °C.The medium was then replaced with the

Wound healing assay
Following a previously described protocol 23 , wounds were created in the HeLa cell culture at 90% confluency using a 100 μl pipette tip.The cells were then cultured in DMEM supplemented with 10% FBS, in the presence or absence of naringenin at concentrations of 100 μM, 200 μM, and 0 μM for 24 h.Photomicrographs of each well were taken under a Leica DM2500 microscope (Germany).

Cell invasion detection
Transwell chambers(Coring, USA) were utilized to detect cell invasion.Filters were coated with 50 mg/ml Matrigel (BD, USA) solution.HeLa cells were inoculated into the upper compartment of the transwell chambers (24 wells; 8 mm pore) and permitted to invade the lower chambers in a DMEM medium fortified with 20% FBS, in the presence or absence of naringenin for 24 h, the invading cells were fixed, stained, and quantified under a microscope.

Statistical analysis
Data analysis was performed with GraphP ad Prism7.0(La Jolla, CA, USA).Data were expressed as mean ± SD.P < 0.05 is considered as the significant difference.

Result Analysis of predicted targets
Naringenin, an active compound in Chinese herbal medicine, is known for treating a variety of diseases.To elucidate the potential mechanism of naringenin in the treatment of cervical cancer, we devised a series of experiments for analysis (Fig. 1).We obtained 287 potential targets of naringenin that were predicted by Pharmmapper.

Targets related to cervical cancer
We downloaded three sets of cervical cancer data (GSE9750, GSE138080, GSE7803) from the GEO database to procure targets related to cervical cancer, resulting in a collection of 967 differential genes of cervical cancer (Fig. 2A).Further, the Genecards database yielded 1741 related targets.We combined the differential genes from GEO with the disease targets from the Genecards database, removing duplicates, which resulted in 2560 disease targets for cervical cancer (Fig. 2B).

PPI network construction
For a more detailed analysis of potential targets, we extracted 85 common targets from potential targets from naringenin and those relevant to cervical cancer (Fig. 2B, C).To construct a "compound-targets" network, these 85 potential working targets were utilized and analyzed via Cytoscape.The resulting PPI network comprised 577 edges and 82 nodes (Fig. 3A), with the top 20 hub targets including AKT1, EGFR, CASP3, SRC, ESR1, MMP9, MMP2, RHOA, PPARG, MAPK14, among others (Fig. 3B).These results propose these targets as potential naringenin targets for cervical cancer treatment.

Functional analysis of candidate targets
To comprehend the biological characteristics of the target gene, we performed a GO function enrichment analysis on the 85 targets, categorizing them into Cell Compounds, Biological Processes and Molecular Functions.Cell Compounds encompassed 67 terms, primarily enriched in membrane raft, membrane microdomain, cell leading edge, focal adhesion, leading edge membrane, cell-substrate junction, receptor complex, protein kinase complex, and more (Fig. 4A).Molecular Functions comprised 105 terms, predominantly enriched in kinase activity, transcription factor binding, nuclear receptor activity, kinase binding, among others (Fig. 4B).Biological Processes covered 1379 terms, mainly focusing on regulation of kinase activity, epithelial cell migration, epithelium migration, epithelial cell differentiation, regulation of 3-kinase signaling, and more (Fig. 4C).

Molecule docking
We utilized Auto Dock Vina for molecular docking to confirm whether the top 10 protein targets interact with naringenin.The results suggested a high binding energy between naringenin and the top 10 targets, particularly with MMP9 (Table 1).As per the molecular docking diagrams, naringenin forms stable hydrogen bonds with amino acid residues ILE-290, THR-211, ASN-204, ASN-54 in the protein structure of AKT1; ARG-94 in the protein structure of EGFR; MET-421, HIS-524, ARG-394 in the protein structure of ESR1; and ARG-249 in the protein structure of MMP9 (Fig. 5).These results suggest that MMP9, EGFR, ESR1, AKT1 could potentially be targets for naringenin.

Impact of naringenin on morphology and viability of Hela cells
To evaluate the influence of naringenin on cervical cancer, we assessed its effect on Hela cell morphology and viability.Following 24 h of incubation with different naringenin concentrations, HeLa cell morphology was recorded under a microscope.Our data indicated that naringenin triggered morphological changes in cells, including shrinkage, rounding, and apoptosis (Fig. 6A).It also diminished HeLa cell viability (Fig. 6B).These results suggest that naringenin may inhibit cellular proliferation in cervical cancer.

Impact of naringenin on HeLa cell migration and invasion
The wound healing assay was utilized to assess the impact of naringenin on HeLa cells migration.Post 24 h of treatment, the HeLa cells' migration distance in the control group was significantly less than that in the treatment group (Fig. 7A), suggesting a substantial inhibitory effect of naringenin on HeLa cell migration.Moreover, the Transwell invasion assay indicated that naringenin significantly reduced the number of invading cells in a dose-dependent manner, compared to the control group following pre-incubation with increasing naringenin concentrations for 24 h (Fig. 7B).This evidence suggests that naringenin may inhibit the migration and invasion of cervical cancer cells.

Naringenin suppression of the EGFR/PI3K/AKT/mTOR pathway
To investigate the molecular mechanisms of naringenin in cervical cancer progression, we conducted a study on the protein expression levels of MMP9, EGFR, AKT and caspase-3 in Hela cells exposed to various naringenin concentrations according to our KEGG enrichment analysis and molecular docking results.Additionally, we detected the levels of EGFR downstream proteins, PI3K and mTOR.The results showed that naringenin decreased the expression levels of MMP9 (Supplementary Fig. 1D), EGFR, PI3K, p-PI3K, p-AKT, and mTOR proteins, while enhancing the expression levels of caspase-3 (Fig. 8, original blots were presented in Supplementary Fig. 1).
These findings indicate that naringenin can exert an anti-cervical cancer effect by suppressing the EGFR and its downstream PI3K/AKT/mTOR.

Discussion
Cervical cancer, now recognized as the fourth most prevalent cancer among women, manifests a high incidence and mortality rate 25,26 .Traditional chemotherapy treatments are often marred by issues of drug resistance, diminishing their efficacy over time.There's a growing interest in Traditional Chinese Medicine (TCM), especially in its use for cancer treatment 27,28 .Derived from natural sources, herbal medicines offer advantages over Western www.nature.com/scientificreports/pharmaceuticals, such as greater efficacy and fewer side effects 29,30 , which underscores the pressing need to identify biologically active TCM-derived ingredients for cervical cancer treatment.Network pharmacology is an emerging discipline rooted in bioinformatics and computer science 31 .This method deviates from the conventional "one drug, one target" strategy, aiming instead to investigate "multiple targets and multiple pathways" inherent in TCM formulas.This approach unravels the relationship between drugs and cancers, and providing a new perspective on drug effects 32 .Many studies suggested that the network  www.nature.com/scientificreports/www.nature.com/scientificreports/pharmacology is a useful tool to uncover the pharmacological mechanisms of TCM 33,34 35,36 .Naringenin, a prominent flavonoid abundant in fruits and Chinese herbal medicines, plays a significant role in disease treatment, particularly in tumorigenesis 37 .Naringenin has been found to induce apoptosis and arrest cell cycles in breast cancer 38 .Additionally, it also reduces the expression of proteins to inhibit proliferation, adhesion, invasion and migration in gastric cancer 39 .Interestingly, recent research also suggests that naringenin possesses inhibitory effects against COVID-19 16 .
In this study, we leveraged network pharmacology and experimental validation to elucidate the mechanism of naringenin in cervical cancer.By accessing the GEO database, we identified 2560 differential genes (DE) from GSE9750, GSE7803, and GSE138080, revealing 85 potential naringenin targets against cervical cancer through PPI and GO and KEGG enrichment analyzes.Several potential targets with cell migration were linked by analysis of GO enrichment, a prediction that we subsequently confirmed using the transwell.Eventually, we selected the top 10 hub targets (AKT1, EGFR, CASP3, SRC, ESR1, MMP9, MMP2, RHOA, PPARG, MAPK14) for molecular docking.All these targets bound to the naringenin molecule, suggesting potential avenues for naringenin-based therapeutics.
The MMP9 gelatinase, a crucial member of the MMP family, contributes significantly to the pathogenesis and progression of numerous cancers.MMP-9 influences the migration and invasion of diverse cancer cells including breast cancer 40 , ovarian cancer 41 , glioblastoma 42 and liver cancer 43 , among others.Zhang et al. 44 found that celastrol hampers proliferation, invasion and migration by regulating MMP-9 in HeLa cells, which was consistent with our results.Thus, it is plausible that naringenin could regulate MMP9 to reduce cell invasion and migration.
The pivotal role of caspase-3 is known during apoptosis and tumor growth.Koeppen et al. 45 revealed that ERBB1 hinders caspase-3 to diminish apoptosis.Lin et al. 46 illustrated that the enhancement of caspase-3 activation promotes apoptosis in NSCLC.Additionally, another study by Lin et al. 47 showed that taxol facilitates apoptosis by modulating caspase-3 in nasopharyngeal carcinoma.Previous studies have underscored caspase-3 as an important target for naringenin treatment.For instance, Bao et al. 48reported that naringenin inhibits proliferation, migration, invasion, and induces apoptosis through AKT pathway in gastric cancer.Bulzomi et al. 49 also found that naringenin can activate caspase-3 in breast cancer.These findings align with our results.
Epidermal growth factor receptor (EGFR) possesses tyrosine kinase activity and is frequently overexpressed in tumors 50 .EGFR can activate downstream signaling pathways, thereby promoting tumor cell proliferation, migration, and invasion through ligand binding 51,52 .Previous studies have shown that EGFR is often overexpressed and associated with migration and invasion in cervical cancer 53 .Our study demonstrates that naringenin effectively inhibits the expression of the EGFR-mediated PI3K/AKT/mTOR signaling pathway, which is consistent with previous findings.The PI3K/AKT/mTOR is an important pathway downstream of EGFR signalling 54 .The PI3K/ AKT/mTOR signalling is known to play an essential part in proliferation, migration, and invasion of cancer cells and presents a potential therapeutic biomarker 55 .Previous studies demonstrated that PI3K/AKT/mTOR pathway is a vital way to treat diseases in traditional Chinese medicine.For instance, Yang et al. 56 reported that PI3K/AKT /mTOR pathway is a significant target for natural medicines on TNBC.Farhanet al. 57 discovered that Artemisinin regulated migration and invasion of uveal melanoma through PI3K/AKT/mTOR pathway.Our study found PI3K/ AKT pathway to be the most significantly enriched pathway, and the subsequent WB experiment demonstrated that naringenin suppresses the activation of PI3K/AKT/mTOR pathway.Thus, this study shows that naringenin could inhibit proliferation, migration, and invasion of HeLa cells through EGFR/PI3K/AKT/mTOR pathway.
In summary, we demonstrated the potential molecular mechanism of naringenin in treating cervical cancer through network pharmacology and molecular docking, providing fresh insights into its mechanism against cervical cancer.Our integrated analysis of databases revealed that MMP-9, EGFR, AKT, and caspase-3 may be the drug targets of naringenin in cervical cancer.Our cellular experiments showed that naringenin can inhibit the proliferation, migration, and invasion of cervical cancer cells, and further western blot confirmed that naringenin achieves these effects by EGFR/PI3K/AKT/mTOR pathway.Additionally, we also discovered that naringenin can modulate other signaling pathways to exert anti-cancer effects, and these other pathways warrant further investigation.In order to further explore the potential clinical utility of naringenin, future studies should include more extensive cellular experiments and in vivo animal studies.

Conclusion
In this study, through network pharmacology and experimental evidence, we have demonstrated that naringenin exerts therapeutic effects on cervical cancer via multiple mechanisms, including the inhibition of proliferation, migration and invasion through the regulation of the EGFR/PI3K/AKT/mTOR pathway.The overview of this study is shown in Fig. 9.This study introduces a novel approach to understanding the effects of naringenin on cervical cancer, providing theoretical support for new drugs for the treatment of cervical cancer.

Figure 1 .
Figure 1.Flowchart of the research process.

Figure 2 .
Figure 2. Predictive targets of naringenin and cervical cancer.(A) Identification of differentially expressed genes (DEGs) in cervical cancer versus normal cervical tissues.Downregulated genes are represented in blue and upregulated genes in red.(B) The Venn diagram illustrating common targets in cervical cancer and naringenin.(C) Targets of naringenin relevant to cervical cancer.

Figure 4 .
Figure 4. Enrichment analysis of GO and KEGG pathways.(A-C) Top 20 of GO enrichment analysis for potential targets.(D) Top 20 "target-pathways against cervical cancer" network.

Figure 6 .Figure 7 .
Figure 6.Impact of naringenin on morphology and viability of HeLa cells.(A) Microscopic imaging of cell morphology at 200× magnification after incubation with varied concentrations of naringenin for 24 h.(B) Viability rates assessed by the CCK-8 assay.Data from three independent experiments are presented as means and standard deviations.*p < 0.05.

Table 1 .
Docking scores of naringenin with corresponding potential targets.
. For instance, Wu et al., and Zhou et al., employed network pharmacology and experimental validation to elucidate the pharmacological mechanisms of Xihuang Pills in prostate cancer and Sijunzi Decoction in colorectal cancer, respectively